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Abstract:

The invention relates to a truncated form of the HIV-1 p17 protein,
designated as AT96, which consists of the residues from 1 to 96 of the
full-length p17 protein, as well as the corresponding nucleic acid
sequences. The truncated AT96 protein has the same immunological features
as the full-length p17 protein but at the same time is devoid of the
typical detrimental biological activities of the HIV-1 p17 protein.

12. The truncated protein according to claim 11, wherein the P17 cell
kinase phosphorylation inhibition activity is tested by assessing
phosphorylation of ERK 1/2 and pAKT in Raji cells.

13. The truncated protein according to claim 11, wherein the truncated
protein has the amino acid sequence of SEQ ID NO:2.

14. A medicament comprising the truncated protein according to claim 11.

15. The medicament of claim 14, wherein the medicament is capable of
evoking an immune response that neutralizes a biological activity of the
HIV p17 protein.

16. The medicament according to claim 15, wherein the medicament is
suitable for treatment or prevention of a HIV infection.

17. A nucleic acid encoding the truncated protein according to claim 11.

18. The nucleic acid according to claim 17 having the nucleotide sequence
of SEQ ID NO:1.

19. A pharmaceutical composition comprising a pharmaceutically effective
amount of the truncated protein according to claim 11.

20. The pharmaceutical composition of claim 19, wherein the composition is
an immunogenic or vaccine composition.

21. The pharmaceutical composition of claim 19, wherein the truncated
protein has the amino acid sequence of SEQ ID NO:2.

22. A method to treat HIV in a subject, the method comprisingadministering
to the subject a therapeutically effective amount of the truncated
protein of claim 11.

23. The method of claim 22, wherein the truncated protein has the amino
acid sequence of SEQ ID NO:2.

Description:

[0001]The present invention generally falls within the immunology field
and in particular relates to a truncated form of the HIV p17 protein that
is particularly suited to be used as a vaccine against the HIV-1 virus.

[0002]The development of an effective anti-HIV-1 vaccine represents one of
the main objectives for the worldwide scientific community. Such a
vaccine must primarily be able to evoke both a significant cell response
and the development of a vigorous humoral activity, the latter
characterized by the production of antibodies capable of neutralizing the
infectivity of the virus.

[0003]In these last years, the attention of the scientific world has been
directed to virus-derived polypeptides to be used as a basis for the
design of antiviral vaccines.

[0004]The p17 protein, which composes the matrix of the HIV-1 virus,
represents a biological target that is particularly interesting from such
a point of view. In fact, even though it has been classified as a
structural protein, it is now clear that p17 performs several functions
in many phases of the viral replication cycle, from the entry of the
virus into the cytoplasm to the integration of the viral genetic material
into the cellular one up to the assembly of new virus particles, thus
playing a role of primary importance not only in the virus architecture
but also in the HIV-1 replication cycle.

[0005]Recent studies have enabled to point out that p17 has a structure
very similar to that of gamma-interferon, a human pro-inflammatory
cytokine. Moreover, cell biology studies have enabled to point out the
ability of p17 to modulate the biological activity of several immune
system cells, both from the native and adaptive compartments. In
particular, the activity of p17 on the CD4.sup.+ T lymphocyte
subpopulation, the preferential target for HIV-1, has showed that this
viral protein is able to stimulate the lymphocyte cells by activating
them and thus making them more susceptible to viral infection.
Furthermore, p17 is able to induce the activated T lymphocytes to release
pro-inflammatory cytokines, such as gamma-interferon and tumor necrosis
factor-alpha, into the cell culture microenvironment, thus generating a
favorable environment for the optimum HIV-1 replication.

[0006]The activity of p17 as a "virokine" depends on the direct
interaction of the protein with a specific receptor expressed on the
surface of the target cells.

[0007]The evidence that p17 is able to act, from the biological point of
view, on HIV-1 target cells, by affecting their functionality and
inducing an increase in the HIV-1 replication activity, causes such a
protein to be considered as an excellent target for the establishment of
vaccination strategies against AIDS. An essential prerequisite is that
p17 should be released into the cell microenvironment from HIV-1-infected
cells. Actually, in vitro HIV-1-infected H9 cells release high amounts of
p17 into the cell culture supernatant. Furthermore, recently it has been
proven that p17 is present as a protein deposit in the lymph nodes of
HIV-1 seropositive patients, also in the anti-retroviral therapy (HAART).
It is interesting to note that p17 was detectable in the lymph node
tissue in the absence of viral particles and/or viral genomes. This
suggests that p17, as already demonstrated for other HIV structural
proteins, acts on certain target cells as an exogenous protein,
independently from the presence of the virus. This explains the data
obtained by some researchers which pointed out a connection between the
presence of high levels of anti-p17 neutralizing antibodies and a
significant delay of the acquired immunodeficiency syndrome progression.
As p17 is inside the glycoprotein coat in the viral architecture and as
such not accessible to antibodies, such data were inexplicable before
finding out that p17 is released by the infected cells.

[0008]The International patent application WO03/016337 describes short
peptides isolated from HIV p17, which represent the p17 neutralizing
epitope (residues from 9 to 22) and which are capable of evoking a
neutralizing immune response when administered as a vaccine.

[0009]However, the use of peptides that only represent limited epitopic
regions as the basis for the formulation of vaccines or as molecules
capable of stimulating and directing the humoral immune response shows
some drawbacks, the major one depending on the fact that peptides usually
exhibit a different conformation from that showed by the full-length
protein. As a result, the antibodies produced following the immunization
with such peptides bring about a poor recognition of the native viral
protein and therefore generate a low-efficiency humoral immune response.

[0010]The International patent application WO03/082908 describes the use
of the full-length p17 protein as a vaccine in order to evoke an immune
response capable of neutralizing the immunostimulating activity exerted
by p17 on human cells. However, the use of the full-length protein as a
vaccine shows the drawback that the administered full-length protein is
capable of having the same noxious biological effects as the native viral
protein.

[0011]To overcome such drawbacks, the present invention provides a
truncated form of the p17 protein, designated as "AT96", which exhibits
the same immunogenic features as the full-length p17 protein, but without
showing the typical noxious biological activities of the full-length
protein.

[0012]The truncated AT96 protein of the invention consists of the amino
acids 1-96 of the p17 protein. Preferably, AT96 is encoded by the
nucleotide sequence shown as SEQ ID NO:1 in the Sequence Listing. Still
more preferably, AT96 has the amino acid sequence shown as SEQ ID NO:2 in
the Sequence Listing.

[0013]The truncated AT96 protein of the invention is a very promising
anti-HIV therapy molecule because, as demonstrated in the experimental
section below, even though it is missing the p17 carboxy-terminal end
(that is the amino acids 97-132), which is important for the biological
activity of the native protein, still it retains the immunogenically
important epitopes, namely the epitopes capable of promoting the
neutralizing cell-mediated and humoral immune response.

[0014]The truncated AT96 protein of the invention has been produced by per
se known recombinant DNA methods.

[0015]In short, the AT96 protein was produced by amplifying the nucleotide
sequence encoding for the amino acids 1-96 of p17 and then cloning it
into the prokaryotic pGEX-4T expression vector (GE Healthcare). Such a
nucleotide sequence was obtained by mutational PCR starting from the
sequence encoding for the full-length p17 derived from the BH10 virus
strain (NCBI accession number M15654). The nucleotide sequence of the
full-length p17 used is shown in the Sequence Listing as SEQ ID NO:3 and
the corresponding encoded amino acid sequence is shown as SEQ ID NO:4.

[0016]By using the specially designed mutagenic AT96BamHI and AT96ECORI
primers, the following were created: [0017]a) the BamHI restriction
site upstream of the AT96 encoding sequence, which is necessary for the
subsequent cloning into the prokaryotic pGEX-4T expression vector and
designed to generate a recombinant clone capable of expressing AT96 in
the form of a fusion protein with the Glutathione-S-Transferase (GST)
enzyme encoded by the vector; and [0018]b) the stop codon downstream of
the GAC triplet encoding the amino acid 96 of p17 and the EcoRI
restriction site, which is necessary for the subsequent cloning into
pGEX-4T.

[0019]The sequences of the AT96BamHI and AT96ECORI primers are shown in
the Sequence Listing as SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0020]The amplification reaction (final volume 200 μl) was carried out
by using 20 ng of a plasmid containing the nucleotide sequence of the
full-length p17 as a template. The conditions used for the PCR reaction
were the following: 94° C., 30 sec; 50° C., 30 sec;
72° C., 60 sec for a total of 30 cycles. The amplified product was
purified by using standard protocols (Qiaquick PCR Purification Kit,
Qiagen), then digested with the restriction enzymes BamHI and EcoRI and
finally cloned into the multiple cloning site of the pGEX-4T plasmid,
previously digested with the same restriction enzymes. The obtained
construct (pGEX-AT96) was sequenced by using the Big Dye Terminator
labeling (Applied Biosystems) in conjunction with analysis by sequencer
and capillary chromatography (ABI PRISM® 7700) following the standard
protocol.

[0021]The recombinant DNA technology was also used for the production of
the AT96 protein according to per se known methods.

[0022]The pGEX-AT96 construct (20 ng) was introduced into E. coli (BL21)
by electroporation and the transformed bacteria were selected on plates
containing ampicillin. The presence of the AT96 insert in the selected
colonies was checked by extraction, purification and restriction of the
plasmid DNA.

[0023]The positive colonies were used to set up high-volume (2-4 liters)
production cultures in liquid LB medium with ampicillin. The bacteria
were incubated at 37° C. until reaching an optical density value
corresponding to the beginning of the plateau phase (0.8 AU) and then
induced to produce the protein by the addition of IPTG
(isopropyl-β-thiogalactopyranoside, Sigma Aldrich) up to a final
concentration of 100 mM and at the temperature of 30° C. The IPTG,
by removing the block on the lacZ operon, allows the recombinant protein
to be expressed in high amounts.

[0024]The AT96 protein, produced as a fusion with the GST enzyme
(Glutathione-S-Transferase, the sequence of which is already inserted
into the vectors of the pGEX series), was extracted by sonication of the
bacterial cells and purified by using an affinity column made up by
Glutathione Sepharose 4B beads (GE Healthcare), which, as being extremely
related to the GST enzyme, capture the fusion protein in a selective way.

[0025]AT96 was then separated from GST by proteolytic excision with the
thrombin protease (GE Healthcare). Alternatively, it may be eluted from
the glutathione sepharose matrix by using a buffer containing reduced
glutathione and kept in solution with the aid of chaotropic substances.
The protein was finally subjected to buffer exchange, by dialyzing it
against a sodium chloride solution with a 3500 kDa cut-off cellulose
membrane (Pierce), and was further purified by HPLC (GE Healthcare) with
an ion exchange column.

[0026]The immunogenic features of the truncated protein were checked
experimentally as described in the following experimental section, which
refer to the attached figures, wherein:

[0027]FIG. 1 represents the three-dimensional structure and the amino acid
sequence (SEQ ID NO:2) of the truncated AT96 protein of the invention.
The amino acid sequence given in FIG. 1 is based on the one-letter code,
whereas the one given in the Sequence Listing is based on the
three-letter code.

[0028]FIG. 2 illustrates the Western Blot results that aim at verifying
the responsiveness of the truncated AT96 protein to anti-p17 antibodies.
Following an electrophoresis run on a 15% polyacrylamide gel, the
proteins were transferred onto a nitrocellulose membrane. The proteins
were detected with a purified mouse monoclonal antibody (MB S-3) directed
against the amino-terminal portion of p17. The membrane was developed
with an HRP-conjugated anti-mouse antibody using DAB (diaminobenzidine)
as the substrate. A) and C) p17 (17 KDa); B) AT96-GST (53 KDa) after
elution with reduced glutathione; D) AT96 (11 KDa) after excision with
the thrombin protease.

[0029]FIG. 3 shows the results of a competition ELISA test between p17 and
AT96 performed to estimate the affinity of the proteins for the MBS-3
antibody. The ratio of the binding between the MBS-3 antibody and the p17
and AT96 proteins is calculated indirectly by using the immunocomplex
solutions to carry out a solid-phase ELISA assay in order to detect p17.
AT96 (.tangle-solidup.) showed the same affinity for mAb as the
full-length p17 (.box-solid.).

[0030]FIG. 4 shows the results from experiments concerning AT96 binding to
the p17 receptor expressed on Raji cells and the neutralization of the
protein-receptor interaction. A) Raji cells incubated with an unrelated
protein (GST); B) Raji cells incubated with AT96 (50 ng); C) Raji cells
incubated with AT96 (50 ng) in the presence of serum of a mouse immunized
with a peptide capable of mimicking the amino-terminal portion of p17
(AT20) (1:50), and containing mouse neutralizing anti-p17 antibodies; D)
Raji cells incubated with AT96 (50 ng) in the presence of serum of a
mouse immunized with a peptide capable of mimicking the carboxy-terminal
portion of p17 (CT18) (1:50).

[0031]FIG. 5 shows the humoral response towards AT96 and p17 35 days after
the first immunization. The antibody responses shown are from groups of
mice immunized three times with (A) AT96 at the doses of 1 ( ), 5
(.box-solid.) and 25 (.tangle-solidup.) μg/mouse or with p17 (B) at
the doses of 1 ( ), 5 (.box-solid.) and 25 (.tangle-solidup.)
μg/mouse. (*) Non-immunized mice. Each serum pool was analyzed in
triplicate by ELISA test.

[0032]FIG. 6 shows a comparison between the lymphoproliferative activities
of p17 and AT96. Cells extracted from spleens of mice immunized with p17
or AT96 were cultured and re-stimulated for 7 days with one or the other
protein to evaluate the ability of the proteins to induce clonal
expansion of specific T lymphocytes. The mice were administered with 3
consecutive doses (25 μg/mouse) of p17 (middle bars), AT96 (right
bars) or PBS (left bars) in combination with Freund's incomplete
adjuvant. Two months after the last booster antigen injection, the mice
were sacrificed and the spleens removed for the cell proliferation
studies. The cells were cultured in the presence or absence of p17 or
AT96. The proliferation was estimated on the basis of the incorporation
of tritiated thymidine. The data are representative of 5 experiments
performed with cells derived from different animals.

[0033]FIG. 7 shows the results relating to the specific expansion of mouse
CD8.sup.+ T cells in response to AT96. The mice received three doses of
AT96 at 25 μg/mouse with Freund's incomplete adjuvant. Two months
after the last booster injection, the mice were sacrificed and the spleen
cells were cultured for 7 days in the absence (A) or the presence (B) of
AT96 as the booster antigen. The cells were labeled with an
FITC-conjugated anti-CD8 monoclonal antibody. The cells included in the
gate plotted in A and B represent, by density and size, the lymphocyte
population. The data were analyzed with the CellQuest program and have
been represented as a dot plot. The ratio of CD8.sup.+ T cells (C) in the
AT96-stimulated cultures is shown in the upper right corner.

[0034]FIG. 8 relates to the assessment of the MAP kinase and pAKT
phosphorylation indexes by Western Blot in Raji cells after stimulation
by p17. Identification of the phosphorylated ERK1/2 (A) and pAKT (B) MAPs
in Raji cells untreated with p17 (lane 1) or treated with p17 for a
period of 5 minutes at the increasing concentrations of 100 ng/ml (lane
2), 200 ng/ml (lane 3), 500 ng/ml (lane 4), 1000 ng/ml (lane 5), 2000
ng/ml (lane 6). The examination of the protein amount was carried out by
assessing the total MAPs (C).

[0035]FIG. 9 shows the results of the MAP kinase phosphorylation indexes
obtained by Western blot assessment in Raji cells after stimulation by
AT96. Identification of the phosphorylated ERK1/2 (A) MAPs in Raji cells
untreated with AT96 (lane 1) or treated with AT96 for a period of 5
minutes at the increasing concentrations of 100 ng/ml (lane 2), 200 ng/ml
(lane 3), 500 ng/ml (lane 4), 1000 ng/ml (lane 5), 2000 ng/ml (lane 6).
The examination of the protein amount was carried out by assessing the
total MAPs (B).

EXAMPLE 1

Recognition of AT96 by Anti-p17 Antibodies Capable of Binding to the p17
Neutralizing Epitope

[0036]The AT96 responsiveness to the anti-p17 antibodies capable of
binding to the p17 neutralizing epitope was assessed by immunoenzymatic
(ELISA) and Western blot assays (FIG. 2).

[0037]In order to verify that the conformation of the functional epitopes
at the interaction with the cell receptor is retained in the AT96
protein, a competition ELISA test was established to evaluate the ratio
of the AT96 interaction in solution with the MBS-3 monoclonal antibody,
which recognizes the functional portion of p17 and inhibits the
p17/receptor interaction [1]. The activity of AT96 in liquid phase as a
competitor on the binding between MBS-3 and p17 present in the well
(solid phase) was then compared to that of p17 used as a self-competitor.

[0038]The purified antibody was incubated with increasing concentrations
of the AT96 or p17 protein and then the immunocomplexes obtained were
used for ELISA assessment of the MBS-3 binding activity to the wild-type
p17 fixed to the solid phase (on the bottom of the plate well).
Recombinant p17, derived from the BH10 viral strain, was added to each
well of a polystyrene plate for immunoenzyme assays at the concentration
of 1 μg/ml in 100 μl PBS, the plates were incubated overnight at
room temperature. The antigen-coated plates are designated as detection
plates; whereas the uncoated plates are designated as reaction plates. In
order to minimize the non-specific absorbance of the proteins, 200 μl
of PBS containing 2% BSA (assay buffer) were added to each well of the
detection plate. Both the reaction and detection plates were incubated
for 1 hour at 37° C. The plates were washed with PBS containing
0.1% tween 20 (wash buffer). To each well of the reaction plate, 100
μl of the appropriate dilution of MBS-3 anti-p17 monoclonal antibody
were added, as well as 100 μl of AT96 at concentrations ranging from
0.1 to 25 μg/ml assay buffer, or 100 μl of assay buffer alone.
After a 2 hour incubation at room temperature, 100 μl aliquots were
transferred from the wells of the reaction plate to those of the
detection plate. The detection plates were incubated for 2 hours at room
temperature and then washed four times with the wash buffer. The MBS-3
monoclonal antibody binding in solid phase was detected by adding an
HRP-conjugated anti-mouse antibody.

[0039]From FIG. 3, it can be seen that the affinity of the MBS-3 antibody
for AT96 is identical to that for the full-length p17 protein, confirming
that the three-dimensional structure of the amino-terminal epitope
recognized by the MBS-3 antibody is perfectly retained and in the
truncated AT96 protein of the invention the epitope is exposed. The AT96
protein, by keeping the functional and immunogenic epitopes intact,
should be able to induce, once inoculated into an organism, a significant
cell-mediated and humoral anti-p17 immune response. In this context,
humoral response is intended to mean the generation of antibodies that
neutralize the biological activity of p17, as they are designed to block
the interaction between p17 and the p17 receptor.

EXAMPLE 2

Interaction Between AT96 and p17 Receptor and Neutralization of the
Interaction

Materials and Methods

[0040]Cells: The ability of AT96 to bind the p17 cellular receptor was
assessed on cells from the Raji lineage, that is to say Burkitt's
lymphoma cells that exhibit a high expression of the p17 receptor. The
cells were cultured in RPMI 1640 medium (Gibco BRL) containing 10% FCS
(fetal calf serum), 100 U/ml penicillin, 50 μg streptomycin and 1 mM
L-glutamine.

[0041]Conjugation of AT96 to biotin: The AT96 protein was conjugated to
biotin to allow for the detection thereof by cytofluorometry through
covalent binding to fluorescent streptavidin. The protein was subjected
to buffer exchange through a dialysis membrane (3500 kDa cut-off, Pierce)
against a sodium chloride/sodium bicarbonate solution, pH 8.0. The
protein was then reacted with biotin succinimide ester (BioSPA) for 3
hours at 4° C. and further dialyzed overnight against PBS
(phosphate buffered saline) containing 0.2 M sodium chloride.

[0042]Binding and neutralization tests: The ability of the sera collected
from animals immunized with a peptide that mimics the amino-terminal
portion of p17, designated as AT20 (SEQ ID NO: 7 in the Sequence
Listing), to block AT96 binding to the p17 receptor (p17R) was assessed
by cytofluorometry. The sera of AT20-immunized animals, diluted in PBS
(1:50), were incubated with biotinylated AT96 (50 ng) for 20 minutes at
4° C. Raji cells were treated for 15 minutes at 4° C. with
FcR blocking reagent (Miltenyi Biotech), centrifuged and then
re-suspended in the sera pre-incubated with AT96. Sera from animals
immunized with a peptide that mimics the carboxy-terminal portion of p17,
designated as CT18, were used as a control. The cells were then washed
with PBS and incubated on ice with 100 ng of streptavidin conjugated to
the APC or PE Cy5.5 fluorochrome (Becton Dickinson). The cytofluorometric
analysis of the samples was performed with a FACSCalibur instrument and
the data were analyzed with the CellQuest Pro program (Becton Dickinson).

[0043]Synthetic peptides: The AT20 synthetic peptide consists of the 20
amino-terminal amino acids of the p17 protein, included between the amino
acid positions 9 and 28 (SEQ ID NO: 7). Whereas the CT18 synthetic
peptide consists of the 18 carboxy-terminal amino acids of p17, included
between the amino acid positions 115 and 132 (SEQ ID NO:8). These
peptides were synthesized in the free form and then were conjugated to
the KLH (Keyhole Limpet Hemocyanin) carrier from Primm.

[0044]Immunizing protocol: C57BL/6 mice were immunized by the
intraperitoneal route with AT20-KLH or CT18-KLH emulsified with Freund's
complete adjuvant at the doses of 1, 5 and 25 μg/mouse and boosted,
for 2 consecutive times at a 15 day interval, with the same dose of
immunogen in incomplete adjuvant. The detection of the anti-AT20 and
anti-CT18 antibodies in the sera of the immunized animals was carried out
by ELISA.

[0045]ELISA: The presence of anti-AT20 and anti-CT18 antibodies in the
sera of the immunized mice was assessed by an ELISA test. The wells of an
ELISA plate (Nunc) were covered with 100 ng of AT96 protein overnight at
room temperature in PBS. After 2 washes with the wash buffer (PBS+0.1%
tween), 100 μl of the assay buffer (PBS+2% BSA) were added and
incubated at 37° C. for 1 hour. After 4 washes, 100 μl of
stepwise dilutions of the sera to be tested (from 1:100 to 1:3200) were
transferred into each well. After incubation for 1 hour at 37° C.,
4 washes were performed and 100 μl/well of a 1:1000 dilution of
HRP-conjugated anti-mouse antibody were added. After incubation for 1
hour at 37° C., 4 washes were performed and 100 μl of the
tetramethylbenzidine substrate were added. The chromogenic reaction was
stopped with 100 μl of 2N sulphuric acid per well.

Results

[0046]The experiments so far illustrated have allowed to establish the
integrity of the amino-terminal portion of the AT96 protein, hypothesized
as the region that allows p17 to interact with its cellular receptor. As
a result, AT96 should be able, just like the wild-type p17 protein, to
interact with the receptor on the cell membrane.

[0047]Experimentally, this has been assessed by assaying the binding of
AT96 to B cells from the Raji lineage which express the p17 receptor on
their surface at a high density. The cells were incubated with the
biotin-conjugated AT96 protein for 30 minutes. After several washes, the
binding of biotinylated AT96 to the cell surface was detected by adding
fluorescent streptavidin (conjugated with the APC or PE Cy5.5
fluorochrome).

[0048]FIG. 4 shows that an unrelated biotinylated protein does not bind to
Raji cells (FIG. 4A). Conversely, AT96 is able to bind the cell receptor
on the membrane of Raji cells (FIG. 4B). In fact, the cytofluorometric
analysis shows that the histogram in B, which represents the AT96-labeled
fluorescent cells, has a higher logarithmic scale of light intensity than
the histogram in A, where the cells are labeled with a molecule unable to
bind the surface of Raji cells. As predicted, the binding of AT96 was
detectable on the whole cell population.

[0049]Similarly, the ability of antibodies directed towards a specific
amino-terminal neutralizing epitope, designated as AT20 [2], to block the
in vitro interaction between p17 and the cellular receptor was also
assessed. The AT96 protein was incubated with sera from animals immunized
with the peptide that reproduces the sequence of the amino-terminal
epitope of p17 (AT20) or with sera from animals immunized with a peptide
capable of mimicking a carboxy-terminal sequence of p17 and not existing
in AT96. The AT96/p17 receptor interaction was then detected by using
fluorescent streptavidin as the fluorescent tracer. From the executed
experiments, AT96, just like wild-type p17, resulted to be neutralized,
with regard to its binding activity to the specific receptor, by
antibodies directed towards the amino-terminal portion (FIG. 4C), but not
by those directed towards the carboxy-terminal portion (FIG. 4D). These
data confirm the hypothesis that the interaction between p17 and its
receptor occurs in a highly specific way, possibly through its
amino-terminal end.

EXAMPLE 3

Induction of Anti AT96 Immune Responses

[0050]Recently, it has been demonstrated that p17, as an antigen, is able
to induce neutralizing and cell-mediated humoral immune responses in
animal models [2]. Furthermore, p17 is able to act as a booster antigen
in vitro, inducing proliferation of T lymphocytes taken from animals
previously immunized with the viral protein.

3.1 Induction of the Humoral Response

[0051]Immunizing protocol: The data obtained in the present study, which
was carried out by using the full-length p17 and the truncated AT96
protein of the invention in parallel, point out that the latter is also
capable of inducing neutralizing humoral responses. The immunization was
performed as follows: 30 9-week-old mice were immunized with different
doses of p17 and AT96 administered in the presence of Freund's incomplete
adjuvant. Each group, consisting of 5 mice, was administered by the
intra-peritoneal route with 1, 5 and 25 μg/mouse of p17 or AT96
protein. Subsequently, 2 consecutive booster injections were given at a
15 day interval with the same dose of immunogen. The negative control was
prepared by inoculating PBS and Freund's incomplete adjuvant into the
mice. The immunized mice were bled at 0, 10, 24, 35 days
post-immunization.

[0052]The immunization schedule with p17 and AT96 for Balb/mice is given
in the following Table 1.

[0053]ELISA: The sera were stored at -20° C. The serum reactivity
against AT96 and p17 was tested by solid-phase ELISA, the results of
which are shown in FIG. 5. The ELISA test was performed as follows. The
wells of an ELISA plate (Nunc) were covered with 100 ng of AT96 protein
overnight at room temperature in PBS. After 2 washes with the wash buffer
(PBS+0.1% tween), 100 μl of the assay buffer (PBS+2% BSA) were added
and incubated at 37° C. for 1 hour. After 4 washes, 100 μl of
stepwise dilutions of the sera to be tested (from 1:100 to 1:3200) were
transferred into each well. After incubation for 1 hour at 37° C.,
4 washes were performed and 100 μl/well of a 1:1000 dilution of
HRP-conjugated anti-mouse antibody were added. After incubation for 1
hour at 37° C., 4 washes were performed and 100 μl of the
tetramethylbenzidine substrate were added. The chromogenic reaction was
stopped with 100 μl of 2N sulphuric acid per well.

[0054]Neutralization test: The sera obtained through the immunizing
program were used to perform neutralizing experiments on the interaction
with the p17 or AT96 protein receptor. Raji cells were incubated for 30
minutes on ice with different amounts of biotinylated p17 or AT96, from
10 to 400 ng/ml. Subsequently, the cells were labeled for 30 minutes on
ice with PE-conjugated streptavidin. For the neutralizing experiments,
the Raji cells were incubated with the immunocomplexes obtained by
pre-incubating the biotinylated p17 with sera from immunized (Ab) or
non-immunized (K) animals at a 1:100 final dilution. The inhibition ratio
was calculated as follows: % of receptor-positive cells in K-% of
receptor-positive cells in Ab/% of receptor-positive cells in K. The
Table 2 below shows the neutralization of the AT96/p17R or p17/p17R
interaction by antibodies generated in animals immunized with different
doses of p17. The % inhibition was calculated as indicated above.

[0055]The AT96 protein resulted to be also able to induce, as the
full-length p17, cell-mediated responses in animal models and to act in
vitro as a booster antigen, inducing proliferation of T lymphocytes taken
from animals immunized with the maximum dose of p17 or AT96 (FIG. 6).
Similar results were obtained by stimulating spleen cells from animals
immunized with the two lower doses of p17 or AT96 (data not shown).

[0056]Proliferation experiments: For the execution of the proliferation
experiments, mouse spleen cells were sown into 96-well U-bottom cell
culture plates at 2×105 cells/well and grown in complete
RPMI-1640 medium (containing penicillin, streptomycin and serum) in the
presence or absence of p17 or AT96 (10 μg/ml). 5 Days later, 1 μCi
of tritiated thymidine was added to the cells and these were then
sacrificed after further 18 hours of culture. The data are presented as a
stimulation index (SI), which is defined as the ratio of the amount of
tritiated thymidine incorporated by the cells in the presence of the
antigen (AT96 or p17) and the amount of tritiated thymidine incorporated
by the cells in the absence of the antigen.

[0057]The cytofluorometric analyses of the spleen cell cultures extracted
from animals immunized with 25 μg of AT96 and re-stimulated in vitro
for seven days with the same protein, also show the specific expansion of
both CD4.sup.+ and CD8.sup.+ T lymphocytes (FIG. 7). In short, the
CD8.sup.+ lymphocytes were labeled with a specific monoclonal antibody,
allowing to note that a specific expansion of both the CD4.sup.+ and
CD8.sup.+ lymphocyte populations occurred in the sample boosted by the
antigen (p17 or AT96). By correlating the percentage of CD8.sup.+ cells
in the samples boosted by the antigen with the percentage in the sample
lacking the booster antigen, it has been possible to calculate the
CD8.sup.+ T cell expansion index (EI) specifically ascribable to the
antigen used, p17 or AT96.

EXAMPLE 4

Comparison of the Biological Activity Between p17 and AT96

[0058]The biological functionality of p17 was assessed on Raji cells.
First of all, the ability of p17, subsequent to its binding with the
cellular receptor, to affect the activity of the cell kinases was
estimated, the which cell kinases are enzymes activated through
phosphorylation, which in turn are able to phosphorylate different
cellular substrates, activating them. In particular, the MAPKs (ERK 1/2)
and pAKT were assessed, the cell survival and the inhibition of the
pro-apoptotic processes depending on the activation thereof.

[0059]The biological power of the p17 protein is demonstrated by its
influence on the phosphorylation index of the kinases, also detected at
low doses and after an exposition time of a few minutes. The
phosphorylated kinases in the Raji cells were detected by the Western
blot method using monoclonal antibodies as specific reagents (Santa
Cruz). The Raji cells were stimulated for 5 minutes with different
concentrations (from 25 ng/ml up to 2 μg/ml) of the p17 protein. In
FIG. 8, it is possible to observe that, already at the lowest
concentration, the p17 protein is able to completely inhibit the
phosphorylation of both ERK 1/2 and pAKT.

[0060]Consequent to this important demonstration of the biological
activity of p17 in Raji cells, we similarly proceeded to estimate the
biological activity of the AT96 protein. Surprisingly, unlike what was
revealed with the full-length p17, the AT96 protein proved not to be able
to inhibit the phosphorylation of ERK 1/2 and pAKT, which were still
active (phosphorylated) even at AT96 concentrations of 2 μg/ml (FIG.
9).

[0061]Thus, it can be inferred from the experimental data obtained that
AT96 exhibits the same immunogenic features as the wild-type p17 protein,
evoking both humoral and cell-mediated immune responses that are
qualitatively and quantitatively similar. Also with regard to its
interaction with the cellular receptor, AT96, as the wild-type p17 does,
results to be able to bind the cellular receptor. Nevertheless, AT96 and
the wild-type p17 differ greatly as for the cellular signal that leads to
the kinase phosphorylation-mediated cell activation. Such a difference
implies that the truncated AT96 protein of the invention is substantially
devoid of those cell kinase activating biological activities that
contribute to the activation of pro-apoptotic processes clearly noxious
for the cell. On the contrary, the full-length p17 protein has such
cell-damaging biological activities, which prevents the effective use
thereof in anti-HIV treatment strategies.

[0062]The use of the viral truncated AT96 protein of the invention
represents an extremely valuable and innovatory approach to the
development of therapeutic and vaccine strategies against AIDS. Given the
great importance of the matrix p17 protein in the biology of the virus,
this protein represents a biological target of great interest.

[0063]The removal of the carboxy-terminal portion of p17 caused no
structural alterations in the immunogenic epitopes. Moreover, the AT96
protein was shown to maintain the ability to interact with the cellular
receptor.

[0064]The experimental evidences gathered during the course of this study
have also allowed to extrapolate extremely significant structure-function
features related to the HIV matrix p17 protein: [0065](i) p17 binding
site for the cellular receptor is localized in the amino-terminal
portion; in fact, the AT96 protein, even though it lacks the
carboxy-terminal region comprised between the amino acids 97 and 132, can
interact with the p17 cellular receptor; [0066](ii) the carboxy-terminal
portion is essential to the development of the biological activity of the
molecule, as AT96, unlike the wild-type p17 protein, does not inhibit the
phosphorylation, hence the biological activity, of cell kinases;
[0067](iii) the binding of p17 to the cellular receptor is not in itself
indicative of biological activity. In fact, AT96 binds the receptor but
does not inhibit the phosphorylation of cell kinases. It follows that the
biological activity of the protein is localized in the part that has been
removed, which is probably committed to interact with other functional
cell structures in blocking the kinases.

[0068]On the basis of these findings, it is evident that the truncated
AT96 protein of the invention may be extremely promising for the
development of anti-HIV-1 vaccine strategies designed to evoke specific
neutralizing, cell-mediated and humoral, immune responses without
biological activities that are potentially detrimental to an
HIV-1-infected organism.

[0069]Therefore, the use of the truncated AT96 protein according to the
invention as a medicament, particularly as a medicament designed to evoke
an immune response that neutralizes the biological activity of the HIV
p17 protein, and thus suitable for use in the treatment or prevention of
HIV infections, falls within the scope of the present invention.

[0070]To this end, the truncated AT96 protein of the invention may be
manufactured in the form of a pharmaceutical composition, preferably an
immunogenic or vaccine composition, comprising a pharmaceutically
effective diluent or carrier and optionally an adjuvant. The immunogenic
or vaccine pharmaceutical composition of the invention is administered
through any suitable administration route including--without any
limitation--the intravenous, subcutaneous, intramuscular, nasal, mucosal
routes, and so on. The selection of the types and amounts of excipients,
diluents, adjuvants, and carriers, which may be selected depending on the
specific administration route chosen, falls within the abilities of the
person of skill in the art. The immunogen dose in the pharmaceutical or
vaccine composition of the invention also varies depending on several
factors and the determination thereof falls within the abilities of the
person of skill in the art, also taking into account the indications
provided in the experimental part of the description. It is however
possible to contemplate a dose comprised within the range of 1-200 μg,
preferably 10-100 μg.